This invention is in the field of encapsulation, in particular methods and apparatus for encapsulation of liquid droplets. The methods of the invention employ electrostatic atomization to form a compound droplet from at least two miscible fluids. The compound droplet comprises a core of a first fluid and a layer of a second fluid completely surrounding the core. The first fluid contains an agent to be encapsulated and the second fluid contains an encapsulating agent.
Encapsulation is used in a variety of well-known applications such as scratch-and-sniff perfumes, carbonless copy paper, laundry detergent, packaged baking mixes, and pharmaceutical drugs for taste masking and sustained release. Commercial techniques for encapsulation include complex coacervation, co-extrusion, interfacial polymerization, desolvation and various coating techniques. Since the 1930s, a number of variations to the emulsion encapsulation technique have evolved. Mathiowitz et al (U.S. Pat. No. 6,143,211) describe a process for preparing microparticles through phase inversion.
Several encapsulation methods involve flow of two liquids through concentric orifices to form a droplet within a droplet, which can also be called a compound droplet. Merrill et al. (U.S. Pat. No. 2,275,154) describes compound droplets formed by gravity. Raley et al. (U.S. Pat. No. 2,766,478) describe compound droplets formed by a combination of gravity and a slight pneumatic pressure. Somerville describes compound droplets formed by a centrifugal encapsulating apparatus (U.S. Pat. No. 3,015,128 and U.S. Pat. No. 3,310,612). Somerville also describes compound droplets formed by extruding concentrically arranged fluid rods of film and filler material into a stream of carrier fluid. The speed of the carrier fluid is selected to cause the rod to elongate and break up into segments which form into individual particles (U.S. Pat. No. 3,389,194). Ganna-Calvo describes methods for manufacturing coated droplets in which a gas focuses two concentrically positioned immiscible streams to a stable unified jet which flows out of the chamber exit orifice and breaks up into coated particles (U.S. Pat. No. 6,405,936). Methods for forming compound droplets from two immiscible or poorly miscible fluids have also been described where the droplets are formed by dissociation of stable electrified coaxial jets (WO 02/060591, Loscertales. I. G. et al., March 2002, Science, 295, 1695-1698, and US Patent Application Publication Number 2004/0069632).
Concentric orifice configurations have also been used to form electrosprays for mass spectrometry applications. Mylchreest et al. describe an electrospray ion source in which the liquid sample is sheathed with a sheath liquid (U.S. Pat. No. 5,122,670).
Liposomes encapsulating an aqueous core have been proposed for drug delivery and gene therapy. The ability of phospholipids to self-assemble into bilayers enclosing an aqueous core (“liposomes”) was first described by Bangham almost four decades ago (Bangham et al., 1965). The development of liposomes as potential drug delivery vehicles was intensely studied in the 70s and 80s, and several liposome-based products are currently on the market (Gregoriadis, 1995). Of these products, only three consist of water-soluble drugs that are encapsulated within the lipid envelope. Although liposomes have been formulated such that a long circulating half-life is achieved, the encapsulation of drugs within the lipid bilayer can be inefficient. Typical encapsulation procedures involve the rehydration of a dried lipid film with a drug-containing solution such that drug is encapsulated upon vesicle formation. This traditional approach yields encapsulation efficiencies of <10%, with the bulk of the drug remaining outside of the liposome (Semple et al., 2001). The removal of the unencapsulated drug is labor-intensive, costly, and results in substantial losses of both drug and lipid. Encapsulation efficiency can be improved by utilizing a pH gradient and taking advantage of drugs that will partition across the membrane and become entrapped within the acidified interior of the liposome (Lasic et al., 1995). Unfortunately, separation of the unencapsulated drug from the loaded liposomes is still problematic, and this approach is not applicable to macromolecular therapeutics that cannot penetrate the bilayer.
One aspect of gene delivery that has been shown to have a major effect on therapeutic gene delivery in vivo is the maintenance of DNA integrity in physiological fluids. More specifically, it has been demonstrated that the destabilization of non-viral vectors in serum causes the exposure of DNA to nucleases (Li et al., 1999). As a result, DNA is rapidly degraded in the blood, thereby preventing it from providing any therapeutic benefit.
This problem has stimulated interest in identifying lipid formulations that bind very strongly to DNA in order to maintain the therapeutic gene in a complex that is resistant to nuclease degradation (Li et al., 1999). However, studies have clearly shown that gene expression cannot occur unless the bound lipid is removed to allow transcription in the nucleus (Zabner et al., 1995; Pollard et al., 1998). Therefore, the use of cationic lipids to prevent DNase degradation can result in a very stable complex that does not disassociate in the intracellular environment, and ultimately inhibits therapeutic gene expression (Oupicky et al., 2002). In an effort to circumvent this dilemma, some researchers have synthesized cationic vectors possessing chemical linkages that can be degraded in an intracellular environment (Bulmus et al., 2003; Dauty et al., 2001). In this way, the vector remains intact in blood to maintain DNA integrity, but dissociation is aided by enzymatic degradation within the cell to allow gene expression.
The problems with encapsulating negatively-charged macromolecules within traditional liposomes stimulated Felgner et al. (1987) to utilize cationic liposomes in an effort to improve the efficiency of DNA encapsulation. This landmark study revolutionized gene delivery, and stimulated the use of cationic lipids in synthetic vectors. However, subsequent studies have clearly shown that true encapsulation is rarely achieved under these conditions, but that an ionic interaction of the DNA with the cationic liposomes causes the formation of a lipid-DNA complex that is ultimately responsible for gene delivery.
Another factor that must be considered when administering vectors in vivo is the interaction with various components in physiological solutions. For example, it is well known that non-viral vectors bind with serum proteins upon IV injection (Yang and Huang, 1997; Dash et al., 1999; Faneca et al., 2002; Opanasopit et al., 2002; Trubetskoy et al., 2003). Furthermore, it has been shown that the binding of serum components causes aggregation in vivo, which decreases the circulation half-life of the vector (Dash et al., 1999; Oupicky et al., 2002). Some studies have taken advantage of the vector aggregation to enhance gene delivery to the lung (Li et al., 1999; Barron et al., 1999; Li and Huang, 2000; Liu and Huang, 2002), but safety concerns and the inability to target other tissues limit the potential applications of this approach. Other researchers have attempted to incorporate high levels of steric stabilizers and targeting ligands to prolong circulating half-lives (Choi et al., 1998; Fajac et al., 1999; Ogris et al., 1999; Tam et al., 2000; Oupicky et al., 2002). Although this approach has been successfully utilized for liposome-based pharmaceuticals and appears to be effective at increasing circulation lifetimes (Papahadjopoulos et al., 1991; Torchilin et al., 1994), studies have also shown that the incorporation of polyethylene glycol (PEG)-conjugated components into vectors disrupts normal cellular processing and ultimately reduces transfection rates (Harvie et al., 2000).
Polymeric vesicles formed from amphiphilic polymers have also been proposed as drug delivery vehicles. Amphiphilic polymers proposed for polymeric vesicles include diblock copolymers of polyethyleneoxide-polyethylene (Discher, B. M. et al. (1999) “Polymerosomes: Tough Vesicles Made from Diblock Copolymers”, Science, 284: 1143-1146), carbohydrate-based polymers based on chitosan (Uchegbu, I. F. et al., (1998) “Polymeric chitosan based vesicles for drug delivery. J. Pharm. Pharmacol., 50, 453-8) and amino acid based polymers (Uchegbu, I. F. et al., (1998) “Polymeric vesicles from amino acid homopolymers”, Proc. Intl. Symp. Control. Rel. Bioact. Mater. 25, 186-7).